The present invention relates to the identification of two novel phosphorylation sites of myosin light chain 1 (MLC1). Phosphorylation of MLC1 at these sites was demonstrated to increase in vivo following pharmacologic preconditioning with adenosine. Monitoring MLC1 phosphorylation provides a useful means for identifying new cardiac or skeletal muscle protective agents, monitoring the extent of preconditioning of cardiac and skeletal muscle tissue, and monitoring the status of a subject with cardiac or skeletal muscle damage. Further, altering MLC1 phosphorylation serves as a means for changing contractility of skeletal and cardiac muscle tissue and for protecting skeletal and cardiac muscle tissue from damage caused by conditions and/or factors including, but not limited to, cardiomyopathies, hypertension, free radicals, ischemia, hypoxia, and ischemia/hypoxia with reperfusion.
Ischemic preconditioning (PC), a phenomenon which exists in all species examined, including humans(Cohen, M. V. and Downey, J. M. Lancet 1993 342:6; Yellon et al. Lancet 1993 342:276-277; Kloner et al. J. Am. Coll. Cardiol. 1994 24:1133-1142), is a form of protection whereby a brief ischemic episode reduces the extent of damage to cardiac and/or skeletal muscle tissue from subsequent prolonged ischemia (Murry et al. Circ. 1986 74:1124-1136). PC may also be recruited pharmacologically, with one of many activators being adenosine, a by-product of adenosine triphosphate (ATP) metabolism (Liu et al. Circ. 1991;84:350-356). Both ischemic and pharmacological PC trigger two windows of protection, the first (classical PC) becoming manifest within 15 minutes and lasting 1-3 hours (Cohen, M. V. and Downey, J. M. Lancet 1993 342:6; Van Winkle et al. Cor. Art Dis. 1991 2:613-619; Li et al. Am. Heart J. 1992 123:346-353; Lawson et al. J. Mol. Cell Cardiol. 1993 25:1391-1402). The short duration of protection afforded by classical PC is likely the result of post-translational protein modifications, as 15 minutes does not suffice to recruit de novo transcription and translation. In contrast, the second window (late or delayed PC), which is manifested 24-72 hours after the conditioning stimulus (Marber et al. Circ. 1993 88:1264-1272; Kuzuya et al. Circ. Res. 1993 72:1293-1299) involves changes in gene expression (Bolli R. Circ Res. 2000 87:972-983) as well as post-translational protein modifications.
While the protective effect of PC is well established, the molecular mechanisms of PC remain elusive. Current research into classical PC focuses primarily on the opening of the inner mitochondrial ATP-sensitive potassium (mitoKATP) channel in response to activation of complex kinase signaling cascades (Cohen et al. Annu. Rev. Physiol. 2000 62:79-109; Marber, M. S. Circ. Res. 2000 86:926-931). Ischemia-induced release of adenosine, bradykinin, opioids, and free radicals leads to receptor-mediated activation of protein kinase C (PKC) (Cohen et al. Annu. Rev. Physiol. 2000 62:79-109; Marber, M. S. Circ Res. 2000 86:926-931). Kinases downstream from PKC that have been implicated in PC include a tyrosine kinase, and a number of mitogen-activated protein kinases (MAPKs), the most likely candidates of which are in the c-Jun N-terminal kinase (JNK) and p38 MAPK families (Cohen et al. Annu. Rev. Physiol. 2000 62:79-109). A key downstream effect of this cascade appears to be the opening of mitoKATP channels, as pharmacological channel opening mimics genuine ischemic PC, and mitoKATP channel blockers abolish cardioprotection (Grover, G. J. J. Cardiovasc. Pharmacol. 1994 24:S18-S27). The metabolic protective effects of channel opening may result from a reduction in ATP hydrolysis (Garlid et al. Circ Res. 1997 81:1072-1082) and an influx of Ca2+ into the mitochondria (Holmuhamedov et al. FASEB J. 1999 13:A1079(Abstr)). PC has also been demonstrated to protect against functional myofilament changes of stunned myocardium in rat trabeculae (Perez et al. Cardiovasc. Res. 1999 42:636-643), thus implying that modification to myofilament proteins may also potentiate protection.
An object of the present invention is to provide methods for identifying agents which protect cardiac, skeletal and smooth muscles from damage via their ability to increase MLC1 phosphorylation in the muscle tissue.
Another object of the present invention is to provide methods and compositions for protecting cardiac and skeletal muscles from damage by increasing phosphorylation of MLC1 in the muscle tissue.
Another object of the present invention is to provide methods and compositions for altering the contractility of cardiac and skeletal muscles by modulating MLC1 phosphorylation in the muscle tissue.
Another object of the present invention is to provide methods for monitoring the phosphorylation status of MLC1 in a subject. Such methods are useful in evaluating whether or not a subject is adequately protected from damage to cardiac and skeletal muscles caused by conditions and/or factors such as cardiomyopathies, hypertension, free radicals, ischemia, hypoxia, and ischemia/hypoxia with reperfusion. In addition, the status of MLC1 phosphorylation is useful in assessing the status of myocardial damage in a subject.
Yet another object of the present invention is to provide methods for identifying kinases and/or phosphatases that act on MLC1 as therapeutic targets for agents that modulate or protect against damage to cardiac and skeletal muscles.